The structural diversity of carbohydrates and their crucial role in many biological functions has prompted the development of new techniques for their characterization. Tandem mass spectrometry has been shown to be a powerful means of getting structural information on sequence, branching and interglycosidic linkages in native or derivatized glycans. Permethylated oligosaccharides have been analyzed by matrix-assisted laser desorption/ ionization (MALDI) using a reflectron time-of-flight mass spectrometer in the post-source decay (PSD) mode. A systematic study of a series of permethylated oligosaccharides has been carried out to formulate a rationale for the fragmentation processes. PSD mass spectra of the sodiated or lithiated molecular species showed intense signals reflecting the preferential cleavage of the glycosidic bonds at N-acetylhexosamine residues. These fragments allow immediate identification of HexNAc units and their location within the sugar sequence. More gene rally, cleavage of the glycosidic bonds yielded fragments bearing either the reducing or nonreducing end of the oligosaccharide, thus allowing assignment of sequence and branching. Glycosylation at position 3 of HexNAc residues led to a highly specific fragmentation process involving the elimination of these substituents and thus allowed unambiguous assignment of this type of interglycosidic linkage. MALDI-PSD mass spectrometry of permethylated oligosaccharides appears to be a valuable complement (and/or alternative) to the techniques currently in use for carbohydrate structural analysis.